The long-term goal of my laboratory is to determine mechanisms of heparanase in brain metastasis and to target heparanase therapeutically for this devastating disease. Our work has implicated heparanase as a promoter of brain metastasis. In particular, we have demonstrated that HPSE is expressed in brain metastatic breast cancer (BMBC) cells and tissues, and functions as a downstream target of HER2/EGFR pathways affecting BMBC cell proliferation. Our objective is now to determine how heparanase regulates BMBC and to use this knowledge to develop new heparanase-based therapies. Underscoring the importance of targeting heparanase, we have made four key discoveries that shed new light on the relevance of this molecule towards the aggressive BMBC phenotype. First, we identified microRNA-1258 as a microRNA that inhibits heparanase and suppresses BMBC. Second, we found that heparanase modulates EGFR phosphorylation at sites which are not targets of lapatinib, a dual HER2/EGFR kinase inhibitor, suggesting heparanase roles in mechanisms of lapatinib resistance. Third, we discovered that HPSE regulates Rac and Rho, critical mediators of cytoskeletal dynamics which is a fundamental process in the interplay between tumor cells and the microenvironment. Fourth, by investigating circulating tumor cells (CTCs) from the blood of BMBC patients, we discovered the expression of heparanase in CTCs, and a significant correlation between its presence, EGFR gene amplification, and ALDH1, a known cancer stem cell marker. A logical next step is to formulate strategies to inhibit HPSE and suppress BMBC. This can be achieved by using miR-1258 as well as new HPSE inhibitors, e.g., non-anticoagulant, glycol-split heparins. One of them, SST0001, has emerged as a potent small-molecule inhibitor of heparanase and is available to us. Based on our discoveries, we hypothesize that heparanase expression and function regulates the cross-talk between BMBC and cells of the brain microenvironment, and initiates multiple effects that are critical for the development and progression of BMBC. By proposing much broader roles for heparanase, which involve enzymatic and non-enzymatic functions, the following aims are designed to identify new mechanisms for this molecule keenly involved in BMBC progression. Aim 1 will determine the mechanisms of heparanase regulation by miR-1258 in relation to BMBC suppression. Aim 2 will identify functions of heparanase inhibitors, SST0001 and miR-1258, and their ability to overcome lapatinib resistance in BMBC cells. Aim 3 will delineate the roles of heparanase during the initial steps of BMBC development, signaling, and in brain-homing CTC modalities. Our approaches will include in vitro and in vivo models, coupled with lentiviral delivery targeting HPSE with miR-1258, new HPSE inhibitors, and cutting-edge CTC technologies. These studies emphasize the strong translational component of our proposed work by providing pre-clinical data to introduce heparanase inhibitors in more effective therapies to treat brain metastasis, in particular brain metastatic breast cancer.